Project Summary The goal of this project is to develop a novel drug, R-4329, for the prevention and/or treatment of Clostridium (C.) difficile pathogenesis. R-4329 is based on a unique protein known as secreted antigen A (SagA) identified from Enterococcus faecium which our research team showed can protect against multiple enteric infections, including C. difficile, by enhancing the integrity of the gut membrane barrier. C. difficile is a ubiquitous anaerobic Gram-positive bacterium that can sporulate and become highly resistant to environment stresses and frontline antibiotics such as clindamycin. Pathogenic strains of C. difficile secret toxins (A and B) that damage intestinal epithelial cells, resulting in inflammatory colitis, severe diarrhea, abdominal pain, flu-like symptoms, and possible death. C. difficile infection (CDI) often occurs following antibiotic-treatment when the endogenous microflora of individuals is altered or severely reduced. Since 2000, the rate of CDI has increased from below 150,000 to over 250,000 cases and has been become one of the most significant hospital-acquired infections with an economic burden of over $1 billion to treat in the USA. Once C. difficile establishes residence in the colon, it is difficult to eradicate with last resort antibiotics (vancomycin) and consequently results in relapses of CDI and inflammatory colitis. An effective approach to treating CDI has been fecal microbiota transplant (FMT) therapy. However, FMT is a heterogeneous and poorly defined therapeutic, for which its safety is still a major concern requiring special FDA approval. New and well-defined therapeutics are desperately needed to prevent and treat CDI. Defined strains of probiotics offer an exciting alternative to prevent and treat CDI and many strains of Lactobacillus (acidophilus, casei, reuteri, plantarum) have been explored as probiotics to inhibit infection of enteric pathogens and mitigate antibiotic-associated diarrhea. Unfortunately, the mechanism of action has been difficult to characterize with limiting beneficial effects in the context of their clinical utility. While more clinical studies are still needed, these recent studies suggest that improved probiotics may be even more effective at controlling CDI. Indeed, our recent results published in the journal Science demonstrate that engineering or ?reprogramming? of probiotics to recombinantly express SagA yields a more protective functionality against enteric infections, including CDI. Our goal is to develop a novel probiotic strain to deliver SagA (referred to as R-4329) as an orally administered drug that functions naturally to induce protective regulatory signals. The key objectives are to: 1) generate L. lactis clones expressing SagA from a genome- integrated operon; 2) select a R-4329 clinical candidate based on growth rate, SagA expression, and stability, and 3) demonstrate R-4329 activity in the murine C. difficile infection model. Successful commercialization of R-4329 will ultimately provide a profound front-line medical advancement in the treatment of enteric infection.